It is well known that leguminous plants under certain conditions "fix nitrogen" directly from the air and convert it to organic nitrogenous compounds, thereby providing nitrogen to the plant for protein synthesis and also enriching the soil around the leguminous plants by leaving nitrogenous nutrients in the soil for later crops. In actual fact, of course, the plants themselves do not fix nitrogen, but the nitrogen fixation occurs in Rhizobium bacteria which exist symbiotically with the legumes in nodules formed in the roots of the plants. Examples of leguminous plants which are capable of symbiotic relationship with Rhizobium bacteria are peas, beans, alfalfa, red clover, white clover, vetch, lupines, and the like.
Other agriculturally important non-leguminous plants, such as grasses and grains, are unable to fix nitrogen directly from the air. Non-Leguminous plants generally depend entirely upon combined nitrogen in the soil, such as nitrates and ammonium salts for nitrogen for protein synthesis. After a series of such crops have been grown on any given field, the combined nitrogen in the soil becomes depleted. Consequently, crop rotation is often practiced, whereby nitrogen-fixing plants are grown in rotation with non-nitrogen-fixing plant to replenish soil nitrogen. Furthermore, some legumes, particularly soybeans, peas, beans and alfalfa, are commercially important as crops themselves, and the growth of these crop plants is greatly facilitated by ample combined nitrogen availablity.
The particular Rhizobium bacteria necessary to a given nitrogen-fixing plant may be universally present in the soil. Different Rhizobium species are adapted to form nodules only in legumes of specific species. Therefore, it is a quite common practice to inoculate the seeds of leguminous plants with an appropriate culture of Rhizobium bacteria. The inoculation can be done by coating the seeds, dusting planted seeds or crops, or by spreading inoculant in the furrows of planted leguminous seeds.
One method of inoculating leguminous seeds is to maintain the Rhizobium bacteria culture in an active living state, by mixing a moist culture of the bacteria with a carrier such as humus or peat. The carrier maintains the bacteria in a moist, living state. However, the shelf life of such live bacterial cultures can be relatively short, because the bacteria die under conditions of storage due to the relative shortage of food and moisture in their environment. An example of a moist type of inoculant mixture is described in U.S. Pat. No. 2,726,948 to Erickson, wherein an active moist bacterial culture is mixed with a mixture of peat, charcoal and limestone, so that water is present to the level of approximately 38% by weight of the whole.
Another method of preparing legume seed inoculants is to convert the bacteria culture to a state of dormancy. One known method used to create dormant bacteria is freeze-drying, as described in U.S. Pat. No. 3,168,796 to Scott, et al. In Scott, Rhizobium bacteria are freeze-dried at temperatures in the range of -35.degree. to -70.degree. C. The dried bacterial cake is then ground and blended with a powdered carrier, which carrier is a non-hydroscopic inert powder less than 40 microns in size. The weight ratio of bacteria to carrier in the Scott patent is in the range of 5 to 400 milligrams bacteria per ounce of carrier. According to the Scott patent, it is critical that water be excluded during the mixing step between bacteria and carrier.
Although freeze-drying Rhizobium bacteria prior to mixing with a carrier usually gives a high initial recovery, the bacteria does not always remain stable for long storage periods. Therefore, it would be advantageous to provide a dry, dormant Rhizobium inoculant which can be easily prepared and which will be stable over fairly long storage times with high yields of viable Rhizobium upon re-exposure to moisture.
Some have suggested that other, non-rhizobium, bacteria may be beneficial to some crop plants. Many terrestrial bacterial species are known and it is quite possible that some may be particularly helpful in the cultivation of field crops because of symbiotic relationships formed between plant and bacteria. In the event such associations are identified, it would become necessary to be able to effectively deliver the bacteria to the field in order to take advantage of this symboisis.